Abstract
After DNAs are damaged, DNA repair proteins accumulate and are activated at the DNA damaged site. These accumulated proteins are visualized as foci by fluorescent immunocytochemistry technique. This allows the DNA damage responses in interphase nuclei to be detected; it was earlier times difficult to analyze DNA damage in situ. In order to analyze DNA damage in interphase cells, either DNA is extracted to assay breaks biochemically, or premature chromosome condensation is conducted to observe as chromatin breaks. Although DNA damage-induced foci are typically analyzed in interphase cells, these foci can be also visualized on mitotic chromosomes. The foci where the repair proteins accumulate at the damage site is observed as mitotic chromosome break site. Since mitotic cells attach loosely or not attached to cell culture vessels, it is difficult to analyze foci on chromosomes in culture vessels under a microscope, so metaphase chromosome spread must be prepared for accurate analysis. The cytocentrifuge system is an ideal method to adhere mitotic cells to microscope slides for the fluorescent immunocytochemistry. This chapter introduces cytocentrifuge method to prepare metaphase spread for DNA damage foci analysis.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Kato TA, Nagasawa H, Weil MM, Little JB, Bedford JS (2006) Levels of gamma-H2AX Foci after low-dose-rate irradiation reveal a DNA DSB rejoining defect in cells from human ATM heterozygotes in two at families and in another apparently normal individual. RadiatRes 166(3):443–453
Genet SC, Fujii Y, Maeda J, Kaneko M, Genet MD, Miyagawa K, Kato TA (2012) Hyperthermia inhibits homologous recombination repair and sensitizes cells to ionizing radiation in a time and temperature dependent manner. J Cell Physiol. https://doi.org/10.1002/jcp.24302
Rogakou EP, Boon C, Redon C, Bonner WM (1999) Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol 146(5):905–915
Rothkamm K, Lobrich M (2003) Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses. Proc Natl Acad Sci U S A 100(9):5057–5062
Cornforth MN, Bedford JS (1983) X-ray-induced breakage and rejoining of human interphase chromosomes. Science 222(4628):1141–1143
Schultz LB, Chehab NH, Malikzay A, Halazonetis TD (2000) p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks. J Cell Biol 151(7):1381–1390. https://doi.org/10.1083/jcb.151.7.1381
Maser RS, Monsen KJ, Nelms BE, Petrini JH (1997) hMre11 and hRad50 nuclear foci are induced during the normal cellular response to DNA double-strand breaks. Mol Cell Biol 17(10):6087–6096. https://doi.org/10.1128/mcb.17.10.6087
Haaf T, Golub EI, Reddy G, Radding CM, Ward DC (1995) Nuclear foci of mammalian Rad51 recombination protein in somatic cells after DNA damage and its localization in synaptonemal complexes. Proc Natl Acad Sci U S A 92(6):2298–2302. https://doi.org/10.1073/pnas.92.6.2298
Gatei M, Young D, Cerosaletti KM, Desai-Mehta A, Spring K, Kozlov S, Lavin MF, Gatti RA, Concannon P, Khanna K (2000) ATM-dependent phosphorylation of nibrin in response to radiation exposure. Nat Genet 25(1):115–119. https://doi.org/10.1038/75508
Davalos AR, Kaminker P, Hansen RK, Campisi J (2004) ATR and ATM-dependent movement of BLM helicase during replication stress ensures optimal ATM activation and 53BP1 focus formation. Cell Cycle 3(12):1579–1586. https://doi.org/10.4161/cc.3.12.1286
Gatei M, Zhou BB, Hobson K, Scott S, Young D, Khanna KK (2001) Ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3 related kinase mediate phosphorylation of Brca1 at distinct and overlapping sites. In vivo assessment using phospho-specific antibodies. J Biol Chem 276(20):17276–17280. https://doi.org/10.1074/jbc.M011681200
Garcia-Higuera I, Taniguchi T, Ganesan S, Meyn MS, Timmers C, Hejna J, Grompe M, D’Andrea AD (2001) Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway. Mol Cell 7(2):249–262. https://doi.org/10.1016/s1097-2765(01)00173-3
Yuan SS, Lee SY, Chen G, Song M, Tomlinson GE, Lee EY (1999) BRCA2 is required for ionizing radiation-induced assembly of Rad51 complex in vivo. Cancer Res 59(15):3547–3551
Siddique MA, Nakanishi K, Taniguchi T, Grompe M, D’Andrea AD (2001) Function of the Fanconi anemia pathway in Fanconi anemia complementation group F and D1 cells. Exp Hematol 29(12):1448–1455. https://doi.org/10.1016/s0301-472x(01)00754-8
Franchitto A, Pichierri P (2002) Bloom’s syndrome protein is required for correct relocalization of RAD50/MRE11/NBS1 complex after replication fork arrest. J Cell Biol 157(1):19–30. https://doi.org/10.1083/jcb.200110009
Stewart GS, Wang B, Bignell CR, Taylor AM, Elledge SJ (2003) MDC1 is a mediator of the mammalian DNA damage checkpoint. Nature 421(6926):961–966. https://doi.org/10.1038/nature01446
Wang B, Matsuoka S, Carpenter PB, Elledge SJ (2002) 53BP1, a mediator of the DNA damage checkpoint. Science 298(5597):1435–1438. https://doi.org/10.1126/science.1076182
Celeste A, Petersen S, Romanienko PJ, Fernandez-Capetillo O, Chen HT, Sedelnikova OA, Reina-San-Martin B, Coppola V, Meffre E, Difilippantonio MJ, Redon C, Pilch DR, Olaru A, Eckhaus M, Camerini-Otero RD, Tessarollo L, Livak F, Manova K, Bonner WM, Nussenzweig MC, Nussenzweig A (2002) Genomic instability in mice lacking histone H2AX. Science 296(5569):922–927
Chan KL, Palmai-Pallag T, Ying S, Hickson ID (2009) Replication stress induces sister-chromatid bridging at fragile site loci in mitosis. Nat Cell Biol 11(6):753–760. https://doi.org/10.1038/ncb1882
Kato TA, Okayasu R, Bedford JS (2009) Signatures of DNA double strand breaks produced in irradiated G1 and G2 cells persist into mitosis. J Cell Physiol 219(3):760–765. https://doi.org/10.1002/jcp.21726
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Kato, T.A. (2023). DNA Damage Foci on Metaphase Chromosomes. In: Gotoh, E. (eds) Chromosome Analysis. Methods in Molecular Biology, vol 2519. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2433-3_10
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2433-3_10
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2432-6
Online ISBN: 978-1-0716-2433-3
eBook Packages: Springer Protocols